Method for measuring membrane potential

ABSTRACT

The invention encompasses an improved method for measuring membrane potential using compounds of the formula I as potentiometric probes. These probes may be used in combination with other fluorescent indicators such as Indo-1, Fura-2, and Fluo-3, such probes may be used in microplate reading devices such as FLIPR™, fluorescent imaging plate reader, sold by Molecular Device Corp., of Sunnyvale, Calif.; flow cytometers; and fluorometers. Such probes are used to measure membrane potential in live cells.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates generally to the fields of biologyand chemistry, and bioanalytical instrumentation. In particular, thepresent invention is directed to composition and methods for use insensing membrane potentials, especially in biological systems.Potentiometric optical probes enable researchers to perform membranepotential measurements in organelles and in cells that are too small toallow the use of microelectrodes. Moreover, in conjunction with imagingtechniques, these probes can be employed to map variations in membranepotential across excitable cells and perfused organs with spatialresolution and sampling frequency that are difficult to achieve usingmicroelectrodes.

[0003] 2. Background of the Art

[0004] The plasma membrane of a cell typically has a transmembranepotential of approximately −70 mV (negative inside) as a consequence ofK⁺, Na⁺ and Cl⁻ concentration gradients that are maintained by activetransport processes. Potentiometric probes offer an indirect method ofdetecting the translocation of these ions. Increases and decreases inmembrane potential (referred to as membrane hyperpolarization anddepolarization, respectively) play a central role in many physiologicalprocesses, including nerve-impulse propagation, muscle contraction, cellsignaling and ion-channel gating (references 1-3). Potentiometric probesare important tools for studying these processes, and for cell-viabilityassessment. Potentiometric probes include the cationic or zwitterionicstyryl dyes, the cationic carbocyanines and rhodamines, the anionicoxonols and hybrid oxonols and merocyanine 540 (references 4-8). Theclass of dye determines factors such as accumulation in cells, responsemechanism and toxicity. Mechanism for optical sensing of membranepotential have traditionally been divided into two classes: sensitivebut slow redistribution of permanent ions from extracellular medium intothe cell, and fast but small perturbation of relatively impeable dyesattached to one face of the plasma membrane (references 2 and 3).

[0005] The bis-barbituric acid and thiobarturic oxonols, often referredto as DiBAC and DiSBAC dyes respectively, form a family of spectrallydistinct potentiometric probes with excitation maxima covering mostrange of visible wavelengths. DiBAC₄(3) and DiSBAC₂(3) have been the twomost popular oxonol dyes for membrane potential measurement (references9 and 11). These dyes enter depolarized cells where they bind tointracellular proteins or membranes and exhibit enhanced fluorescenceand red spectral shifts. Increased depolarization results in more influxof the anionic dye and thus an increase in fluorescence. DiBAC₄(3)reportedly has the highest voltage sensitivity. The long-wavelengthDiSBAC₂(3) has frequently been used in combination with the UVlight-excitable Ca²⁺ indicators Indo-1 or Fura-2 for simultaneousmeasurements of membrane potential and Ca²⁺ concentrations. Interactionsbetween anionic oxonols and the cationic K⁺-valinomycin complexcomplicate the use of this ionophore to calibrate potentiometricresponses. DIBAC and DiSBAC dyes are excluded from mitochondria becauseof their overall negative charge, making them superior to carbocyaninesfor measuring plasma membrane potentials.

[0006] In general, DiBAC and DiSBAC dyes bearing longer alkyl chains hadbeen proposed to have better propertie for measuring membrane potentials(references 5 and 12). DiSBAC₆(3) has been selected to use in aFRET-based membrane potential assay (reference 12). There are no reportson DiBAC₁ and DiSBAC₁ for measuring membrane potentials.

[0007] It has been discovered that DIBAC₁(3) and DiSBAC₁(3) that possessunexpected properties that can be used to measure membrane potentialswith FLIPR and other fluorescence devices. Compared with other membersof DiBAC and DiSBAC family, DIBAC₁(3) and DiSBAC₁(3) give strongersignal and faster response besides its better water solubility.

SUMMARY OF THE INVENTION

[0008] The invention encompasses an improved method for measuringmembrane potential using compounds of the formula I as potentiometricprobes. These probes may be used in combination with other fluorescentindicators such as Indo-1, Fura-2, and Fluo-3, calcium green or Fluo-4.Such probes may be used in microplate reading devices such as FLIPR,fluorescent imaging plate reader,sold by Molecular Device Corp., ofSunnyvale, Calif.; flow cytometers; and fluorometers. Such probes areused to measure membrane potential in live cells.

[0009] The invention also encompasses test kit containing reagents ofcompound I, compound I in combination with a fluorescent reagent and inparticular fluorescent indicators such as Indo-1, Fura-2, Fluo-3,calcium green or Fluo-4.

[0010] Another aspect of the invention involves a method for generatingvoltage sensitive fluorescent changes comprising incubating the membranewith:

[0011] (a) A first reagent selected from the potentiometric probes whichredistribute from one side of the membrane to the opposite side inresponse to transmembrane potential; and a second reagent selected fromthe group consisting of non-fluorescent dyes or pigments that re notmembrane-permeable, and undergo energy transfer with the first reagentone side of the membranes to reduce or eliminate the fluorescence signalon that side; or

[0012] (b) A first reagent selected from the potentiometric probes whichredistribute from one side of the membrane to the opposite side inresponse to transmembrane potential; and a second reagent selected fromthe group consisting of non-fluorescent dyes or pigments that re notmembrane-permeable, and absorb the excitation light or emission from thefirst reagent one side of the membranes to reduce or eliminate theundesired fluorescence signal; or

[0013] (c) A first reagent selected from the potentiometric probes whichredistribute from one side of the membrane to the opposite side inresponse to transmembrane potential; and a second reagent selected fromthe group consisting of fluorescent or luminescent probes which undergoenergy transfer with the first reagent, said second reagent beinglocated adjacent to either the one side or the other side of themembrane.

BRIEF DESCRIPTION OF THE FIGURES

[0014]FIG. 1 is a reaction scheme for making DiBAC and DISBAC.

[0015]FIG. 2 illustrates the voltage-dependent fluorescence intensitychanges of DiSBAC₁(3) in P2X2 cells at different ATP concentrations.

[0016]FIG. 3 illustrates the absorption spectra of DisBAC₁(3),DIsBAC₂(3), DisBAC₄(e), DIBAC₁(3) and DiBAC₄(3) in 1:1 methanol/water.

[0017]FIG. 4 illustrates the fluorescence spectra of DisBAC₁(3),DisBAC₂(3), DisBAC₃(3), DiBAC₄(3) DIBAC₁(3) and DiBAC₄(3) in 1:1methanol/water.

DETAILED DESCRIPTION OF THE INVENTION

[0018] Compounds useful in practicing the present invention are madeaccording to methods described by G. W. Fischer in Chem Ber, 1969,102:2609-2620, as shown in FIG. 1.

[0019] The invention is illustrated by the following examples:

EXAMPLE 1.

[0020] Preparation of DiSBAC₁(3)

[0021] DiBAC and DiSBAC dyes are prepared based on the procedure ofethyl and butyl derivatives) (H. Bartsch and G. Haubold, Arch. Pharm.1982, 315, 761-766). Specifically, malonaldehydebis(phenylimine)monohydrochloride (2.6 g, 10 mmol) and1,3-dimethyl-2-thiobarbituric acid (3.5 g, 20 mmol) are dissolved inacetonitrile (40 mL). To the solution is added triethylamine (2 g, 20mmol). The reaction mixture is refluxed until the starting materials arecompletely consumed as indicated by TLC. The mixture is cooled to roomtemperature, and poured into acidic water (pH 2-3, 350 mL). Theresulting suspension is filtered to collect the solid that is washedwith cold water and air-dried. The crude product is further purified ona silica gel column using a gradient of dichoromethano/metahnol to givethe desired product.

[0022] DiSBAC₁(5), DiBAC₁(3), DiBAC₁(5) and other oxonol dyes areprepared analogous to the above procedure.

EXAMPLE 2.

[0023] Measuring Membrane Potentials Using DiSBAC₁(3) in Combinationwith the Fluorescence Imaging Plate Reader (FLIPR™)

[0024] This specific example illustrates how to use DiSBAC₁(3) in P2X2cells in combination with FLIPR™, fluorescent imaging plate reader soldby Molecular Devices Corp. of Sunnyvale, Calif. P2X2 cells are 1321 N1astrocytoma cells transfected to overexpress the purinergic P2X2ligand-gated ion channel. P2X2 belongs to a class of purinergic ionchannels that pass calcium and sodium in response to purine, includingadenosine 5′-triphosphate (ATP). P2X2 cells are propagated andmaintained in DME (high glucose), 10% FCS, 1× Pen/Strep and 2 mML-glutamine. Doubling time is approximately 36 hours. P2X2 cells shouldbe split at a 1 to 2 ratio upon confluence. The cells should be carriedfor no more than 20 passages. When approaching this limit, a new frozenvial of cells should be resurrected. Following is a typical kitprocedure:

[0025] 1. Plate 40,000 P2X2 cells in 100 μL per well for 96 well platesor 10,000 P2X2 cells in 25 μL per well for 384 well plates forovernight.

[0026] 2. Prepare 1× Loading Buffer.

[0027] 2.1 To prepare the 1× Assay Buffer, pipette 10 mL of 10× ReagentBuffer (1× Hanks' Balance Saline Solution+20 mM HPEPES, pH 7.40) anddilute in 90 mL of distilled water. Adjust pH to 7.4 using 1.0 N NaOHand/or 1.0N HCl.

[0028] 2.2 To prepare 10 mM stock solution of DiSBAC₁(3), dissolve 3.8mg DiSBAC₁(3) in 1 mL DMSO

[0029] 2.3 To prepare 10% pluronic acid, dissolve 400 mg pluronic acidin 4 mL water. Heat in 37° C. to complete dissolution.

[0030] 2.4 To prepare 1× Loading Buffer, add 30 μL of stock DiSBAC₁(3),8 μL 10% pluronic acid, and 20.0 mg DB71 in the 1× FLIPR assay buffer.

[0031] 3. Load Cells with 1× Loading Buffer

[0032] 4. Remove cell plates from the incubator

[0033] 5. Add 100μL of 1× Loading Buffer per well of 96-well plates or25 μL per well of 384-well plates.

[0034] 6. Incubate plates at 37 ° C. for 30 minutes.

[0035] 7. Run the FLIPR Membrane Potential Assay

[0036] 7.1 Make 5× compound plate prior to running the FLIPR assay.Dissolve 27.5 mg of ATP (Sigma Cat# A3377) in 1 mL of sterile water tomake a 50 mM stock solution. Make appropriate dilutions for 100 nM, 1 uMand 10 uM and transfer a minimum of 200 μL to each well of a compoundplate.

[0037] 7.2 Confirm that Membrane Potential Filter is installed. Choosep2x2.fcf for experimental setup in the FLIPR software. Set up theappropriate experiment parameters.

[0038] 7.3 After incubation, transfer the plates directly to FLIPR andbegin the Membrane Potential Assay.

[0039] 8. Run FLIPR and perform data analysis.

[0040] Curve of the ATP dose response should look similar to that shownin FIG. 2; an initial depolarization event depicted as an increase influorescence followed by repolarization or decay in signal nearbaseline. Also, EC50 should be approximately in the 10- 100 nM range.

EXAMPLE 3.

[0041] Measuring Membrane Potentials Using DiBAC₁(3) in Combination witha Microscope

[0042] DiBAC₁(3) is used to measure membrane potential change with amicroscope as described by L. M. Loew (Methods in Cell Biology, vol. 38,pp195-209).

EXAMPLE 4.

[0043] Measuring Membrane Potentials Using DiSBAC₁(3) in Combinationwith Flow Cytometer

[0044] DiBAC₁(3) is used to measure membrane potential change with aflow cytometer as described by L. M. Loew (Methods in Cell Biology, Vol.41, Part A, pp195-209).

EXAMPLE 5.

[0045] Water Solubility and Hydrophobicity Comparison of DiBAC andDiSBAC Dyes

[0046] DisBAC₁(3), DisBAC₂(3), DisBAC₃(3), DisBAC₄(3), DiBAC₁(3) andDiBAC₄(3) are dissolved in DMSO (3 mM). The DMSO stock solutions arerespectively partrioned in 1:1 octanol/water mixture. The concentrationsof the oxonol dyes in octanol and water layers are determined byabsorption spectra. The results are summarized in the following table I.As shown in the table, DisBAC₁(3) and DisBAC₁(3) are much morehydrophilic than the other oxonol dyes. They also have much better watersolubility. TABLE 1 λ_(max) in octanol Absorbance Absorbance inAbsorbance in Water/ Relative Compound (nm): in Water: Octanol:Absorbance in Octanol Values: DiBAC₁(3) 495 1.297 0.3104 4.179 1DiBAC₄(3) 497 0.002501 2.854 0.00088 0.00021 DiSBAC₁(3) 538 0.061470.9126 0.0674 1 DiSBAC₂(3) 543 0.01143 2.187 0.0052 0.078 DiSBAC₃(3) 5440.002256 4.331 0.0005 0.0077 DiSBAC₄(3) 544 0.003318 3.226 <0.0010<0.0077

EXAMPLE 6.

[0047] Absorption Comparison of DiBAC and DiSBAC Dyes

[0048] DisBAC₁(3), DisBAC₂(3), DisBAC₃(3), DisBAC₄(3), DiBAC₁(3) andDiBAC₄(3) are dissolved in methanol (1 mM). The stock solutions arediluted with 1:1 methanol/water, and the absorption spectra are recordedin a spectrophotometer. As shown in FIG. 3, DiBAC₁(3) and DisBAC₁(3)possess unexpected blue shift compared to the other oxonol dyes.

EXAMPLE 7.

[0049] Fluorescence Comparison of DiBAC and DiSBAC Dyes

[0050] DisBAC₁(3), DisBAC₂(3), DisBAC₃(3), DisBAC₄(3), DiBAC₁(3) andDiBAC₄(3) are dissolved in methanol (1 mM). The stock solutions arediluted with 1:1 methanol/water, and the absorption spectra are recordedin a fluorometer. As shown in FIG. 4, DiBAC₁(3) and DisBAC₁(3) possessunexpected blue shift compared to the other oxonol dyes.

EXAMPLE 8.

[0051] Fluorescence Response Comparison of DiBAC and DiSBAC Dyes forSensing Membrane Potentials in FLIPR Assays

[0052] DisBAC₁(3), DisBAC₂(3), DisBAC₃(3), DisBAC₄(3), DiBAC₁(3) andDiBAC₄(3) are dissolved in DMSO (1 mM). The stock solutions arerespectively used to assay membrane potential changes in the P2X2 cellsin combination with FLIPR™ as described in Example 2. The results aresummarized in the following table 2. TABLE 2 Fluorescence enhancement by10 μM Compounds ATP stimulation (in folds) Response speed DiBAC₁(3) 3.7fast DiBAC₄(3) 3.3 slow DisBAC₁(3) 108.5 fast DisBAC₂(3) 38.6 moderateDisBAC₃(3) 14.5 slow DisBAC₄(3) 2.3 slow

[0053] As shown in the table 2, DisBAC₁(3) is much more sensitive, andhas faster response to membrane potential change than the rest DisBACs.DIBAC₁(3) also has much faster response to membrane potential changethan DiBAC₄(3).

EXAMPLE 9.

[0054] Use of DiSBAC₁(3) as a Fluorescent Indicator of TransmembranePotential

[0055] Depolarization of PC 12 Cells

[0056] Protocols for transmembrane potential measurements are summarizedbriefly since they are similar to those given in detail in Example Iabove. The Bis-(1,3-dimethylthiobarbituric acid) trimethine oxonol,[DiSBAC₁(3)], fluorescent reagent may be purchased from Molecular Probes(Eugene, Oreg., USA). The 1× Cell-Loading Buffer for DiSBAC₂(3),consists of sodium-free Tyrode's Buffer (SFTB), 2.5 uM DiSBAC₁(3), and200 uM Direct Blue 71 (as the fluorescence quencher).

[0057] A rat pheochromocytoma (adrenal) cloned cell line, PC12, is grownin RPMI 1640 culture medium with 10% fetal calf serum (FCS), 1%penicillin/streptomycin (PS), 2 mM L-glutamine, and 1 mM sodiumpyruvate. Cells were grown in suspension, and subsequently centrifugedfrom growth medium and resuspended in DiSBAC₂(3)], 1× Cell-LoadingBuffer. Approximately 100,000 cells were plated per well in a 96 wellmicrotiter plate pre-coated with poly-D-lysine to enchance celladhesion, centrifuged at 1000 rpm for 4 minutes, and placed in anincubator for an additional 20 minutes. Cells were not washed with anyliquid medium, nor was the 1× Cell-Loading Buffer removed prior toperforming fluorescence measurements.

[0058] The fluorescently labeled cells were analyzed for changes inmembrane potential by using FLIPR™ fluorescent imaging plate reader.Briefly, cells were depolarized with addition of 75 mM potassiumgluconate in sodium containing Tyrode's Buffer (SCTB). To inhibitvoltage-gated sodium channels cells were previously incubated with 100uM tetrodotoxin (TTX) for 5 minutes prior to depolarization. The datareveal that cell-depolarization (due to potassium addition) causesincreased DiSBAC₁(3) fluorescence. Inhibition of sodium channels by TTXresults in smaller changes in membrane potential upon potassium additionas indicated by a smaller increase in fluorescence as compared to thepositive control (75 mM potassium gluconate without TTX).

[0059] The above examples illustrate the present invention and are notintended to limit the invention in spirit of scope.

REFERENCES

[0060] 1. Zochowski M, Wachowiak M, Falk C X, Cohen L B, Lam Y W, AnticS, Zecevic D., Imaging membrane potential with voltage-sensitive dyes.Biol Bull 198, 1-21 (2000).

[0061] 2. Plasek J, Sigler K, Slow fluorescent indicators of membranepotential: a survey of different approaches to probe response analysis.J Photochem Photobiol B 33, 101-124 (1996).

[0062] 3. Loew L M., Characterization of Potentiometric Membrane Dyes.Adv Chem Ser 235, 151 (1994)

[0063] 4. Wu J-Y, Cohen L B., Fast Multisite Optical Measurement ofMembrane Potential. In Fluorescent and Luminescent Probesfor BiologicalActivity, Mason W T, Ed., pp. 389-404 (1993).

[0064] 5. Loew L M., Potentiometric Membrane Dyes. In Fluorescent andLuminescent Probes for Biological Activity, Mason W T, 2^(nd) Ed. Pp.210-221 (1999).

[0065] 6. Smith J C., Potential-sensitive molecular probes in membranesof bioenergetic relevance. Biochim Biophys Acta 1016, 1-28 (1990).

[0066] 7. Gross D, Loew L M., Fluorescent indicators of membranepotential: microspectrofluorometry and imaging. In Methods Cell Biol 30,193-218 (1989).

[0067] 8. Freedman J C, Novak T S. Optical measurement of membranepotential in cells, organelles, and vesicles. Methods Enzymol 172,102-122 (1989)

[0068] 9. Bronner C, Landry Y. The use of the potential-sensitivefluorescent probe bisoxonol in mast cells. Biochim Biophys Acta 1070,321-331 (1991).

[0069] 10. Shapiro H M. Cell membrane potential analysis. Methods CellBiol 41, 121-133 (1994).

[0070] 11. Loew L M. Confocal microscopy of potentiometric fluorescentdyes. Methods Cell Biol 38, 195-209 (1993).

[0071] 12. Gonzalez J E, Tsien R Y. Improved indicators of cell membranepotential that use fluorescence resonance energy transfer. Chem Biol 4,269-277 (1997).

What is claimed is:
 1. In a method for measuring transmembrane potentialchanges in a biological cell, the improvement comprising using compoundof Structure I as a potentiometric probe.


2. The method of claim 1 comprising: (a) contacting a compound ofstructure I with cell membrane; (b) stimulating membrane potentialchanges physically or with a biologically active substance; and (c)measuring the fluorescence or luminescence changes.
 3. The method ofclaim 1, wherein the potentiometric probe is used to measuretransmembrane potential changes in combination with a second coloredreagent.
 4. The method of claim 1, wherein the membrane is a plasmamembrane of a biological cell.
 5. The method of claim 1, wherein themeasurement is made in a fluorescence microplate reader.
 6. The methodof claim 5, wherein the fluorescence microplate reader is a fluorometricplate reader having an integrated pipeting and fluidic system.
 7. Themethod of claim 1, wherein the measurement is made with a fluorescenceflow cytometer.
 8. The method of claim 1, wherein the measurement ismade in a fluorescence microscope.
 9. A method of claim 1 wherein acompound of Structure I is used in combination with a second fluorescentindicator.
 10. The method of claim 9, wherein the second fluorescentindicator is Indo-1, Fura-2 and Fluo-3, Calcium Green or Fluo-4.
 11. Atest kit for measuring membrane potential changes comprising a compoundof structure I as a reagent.
 12. A test kit according to claim 11 formeasuring membrane potential changes comprising a compound of structureI and a second fluorescent reagent.
 13. A test kit according to claim 11further containing Indo-1, Fura-2, Fluo-3, Calcium Green or Fluo-4. 14.A test kit according to claim 11 for measuring membrane potentialchanges comprising a compound of structure 1 and a secondnon-fluorescent colored reagent.